Adenovirus-mediated gene transfer of inhibitors of apoptosis protein delays apoptosis in cerebellar granule neurons

The inhibitor of apoptosis (IAP) family of antiapoptotic genes, originally discovered in baculovirus, exists in animals ranging from insects to humans. Here, we investigated the ability of IAPs to suppress cell death in both a neuronal model of apoptosis and excitotoxicity. Cerebellar granule neurons undergo apoptosis when switched from 25 to 5 mM potassium, and excitotoxic cell death in response to glutamate. We examined the endogenous expression of four members of the IAP family, X chromosome-linked IAP (XIAP), rat IAP1 (RIAP1), RIAP2, and neuronal apoptosis inhibitory protein (NAIP), by semiquantitative reverse PCR and immunoblot analysis in cultured cerebellar granule neurons. Cerebellar granule neurons express significant levels of RIAP2 mRNA and protein, but expression of RIAP1, NAIP, and XIAP was not detected. RIAP2 mRNA content and protein levels did not change when cells were switched from 25 to 5 mM potassium. To determine whether ectopic expression of IAP influenced neuronal survival after potassium withdrawal or glutamate exposure, we used recombinant adenoviral vectors to target XIAP, human IAP1 (HIAP1), HIAP2, and NAIP into cerebellar granule neurons. We demonstrate that forced expression of IAPs efficiently blocked potassium withdrawal-induced N-acetyl-Asp-Glu-Val-Asp-specific caspase activity and reduced DNA fragmentation. However, neurons were only protected from apoptosis up to 24 h after potassium withdrawal, but not at later time points, suggesting that IAPs delay but do not block apoptosis in cerebellar granule neurons. In contrast, treatment with 100 microM or 1 mM glutamate did not induce caspase activity and adenoviral-mediated expression of IAPs had no influence on subsequent excitotoxic cell death.     

Ethanol induces apoptosis in cerebellar granule neurons by inhibiting insulin-like growth factor 1 signaling

The ability of ethanol to interfere with insulin-like growth factor 1 (IGF-1)-mediated cell survival was examined in primary cultured cerebellar granule neurons. Cells underwent apoptosis when switched from medium containing 25 mM K+ to one containing 5 mM K+. IGF-1 protected granule neurons from apoptosis in medium containing 5 mM K+. Ethanol inhibited IGF-1-mediated neuronal survival but did not inhibit IGF-1 receptor binding or the neurotrophic action of elevated K+, and failed to potentiate cell death in the presence of 5 mM K+. Inhibition of neuronal survival by ethanol was not reversed by increasing the concentration of IGF-1. Significant inhibition by ethanol (15-20%) was observed at 1 mM and was half-maximal at 45 mM. The inhibition of IGF-1 protection by ethanol corresponded to a marked reduction in the phosphorylation of insulin receptor substrate 1, the binding of phosphatidylinositol 3-kinase (PI 3-kinase), and a block of IGF-1-stimulated PI 3-kinase activity. The neurotrophic response of IGF-1 was also inhibited by the PI 3-kinase inhibitor LY294002, the protein kinase C inhibitor chelerythrine chloride, and the protein kinase A inhibitor KT5720, but unaffected by the mitogen-activated protein kinase kinase inhibitor PD 98059. These data demonstrate that ethanol promotes cell death in cerebellar granule neurons by inhibiting the antiapoptotic action of IGF-1.     

Metabolic and genetic analyses of apoptosis in potassium/serum-deprived rat cerebellar granule cells

Cerebellar granule cells maintained in medium containing serum and 25 mM potassium undergo an apoptotic death within 96 hr when switched to serum-free medium with 5 mM potassium. Because large numbers of apparently homogeneous neurons can be obtained, this represents a potentially useful model of neuronal programmed cell death (PCD). Analysis of the time course and extent of death after removal of either serum or K+ alone demonstrated that a fast-dying (T(1/2) = 4 hr) population (20%) responded to serum deprivation, whereas a slow-dying (T(1/2) = 25 hr) population (80%) died in response to K+ deprivation. Taking advantage of the complete death after removing both K+ and serum, changes in metabolic events and mRNA levels were analyzed in this model. Glucose uptake, protein synthesis, and RNA synthesis fell to <35% of control by 9 hr after potassium/serum deprivation, a time when 85% of the cells were still viable. The pattern of the fall in these metabolic parameters was similar to that reported for trophic factor-deprived sympathetic neurons. Most mRNAs decreased markedly after K+/serum deprivation. Levels of c-jun mRNA increased fivefold in potassium/serum-deprived granule cells; c-jun is required for cell death of sympathetic neurons. mRNA levels of cyclin D1, c-myb, collagenase, and transin remained relatively constant in potassium/serum-deprived granule cells. These data demonstrate the existence of two populations of granule cells with respect to cell death and define common metabolic and genetic events involved in neuronal PCD.     

Ornithine decarboxylase activity during development of cerebellar granule neurons

Ornithine decarboxylase (ODC), the key enzyme for polyamine biosynthesis, dramatically decreases in activity during normal cerebellar development, in parallel with the progressive differentiation of granule neurons. We have studied whether a similar pattern is displayed by cerebellar granule neurons during survival and differentiation in culture. We report that when granule cells were kept in vitro under trophic conditions (high K+ concentration), ODC activity progressively decreased in parallel with neuronal differentiation. Under nontrophic conditions (cultures kept in low K+ concentration), the enzymatic activity dropped quickly in parallel with an increased apoptotic elimination of cells. Cultures kept in high K+ but chronically exposed to 10 mM lithium showed both an increased rate of apoptotic cell death at 2 and 4 days in vitro and a quicker drop of ODC activity and immunocytochemical staining. A short chronic treatment of rat pups with lithium also resulted in transient decrease of cerebellar ODC activity and increased programmed cell death, as revealed by in situ detection of apoptotic granule neurons. The present data indicate that a sustained ODC activity is associated with the phase of survival and differentiation of granule neurons and that, conversely, conditions that favor their apoptotic elimination are accompanied by a down-regulation of the enzymatic activity.     

N-Methyl-D-aspartate inhibits apoptosis through activation of phosphatidylinositol 3-kinase in cerebellar granule neurons. A role for insulin receptor substrate-1 in the neurotrophic action of n-methyl-D-aspartate and its inhibition by ethanol

Primary cultured rat cerebellar granule neurons underwent apoptosis when switched from medium containing 25 mM K+ to one containing 5 mM K+. N-methyl-D-aspartate (NMDA) protected granule neurons from apoptosis in medium containing 5 mM K+. Inhibition of apoptosis by NMDA was blocked by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY294002, but it was unaffected by the mitogen-activated protein kinase kinase inhibitor PD 98059. The antiapoptotic action of NMDA was associated with an increase in the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), an increase in the binding of the regulatory subunit of PI 3-kinase to IRS-1, and a stimulation of PI 3-kinase activity. In the absence of extracellular Ca2+, NMDA was unable to prevent apoptosis or to phosphorylate IRS-1 and activate PI 3-kinase. Significant inhibition of NMDA-mediated neuronal survival by ethanol (10-15%) was observed at 1 mM, and inhibition was half-maximal at 45-50 mM. Inhibition of neuronal survival by ethanol corresponded with a marked reduction in the capacity of NMDA to increase the concentration of intracellular Ca2+, phosphorylate IRS-1, and activate PI 3-kinase. These data demonstrate that the neurotrophic action of NMDA and its inhibition by ethanol are mediated by alterations in the activity of a PI 3-kinase-dependent antiapoptotic signaling pathway.     

Neurotrophins rescue cerebellar granule neurons from oxidative stress-mediated apoptotic death: selective involvement of phosphatidylinositol 3-kinase and the mitogen-activated protein kinase pathway

Cerebellar granule neurons maintained in medium containing serum and 25 mM K+ reliably undergo an apoptotic death when switched to serum-free medium with 5 mM K+. New mRNA and protein synthesis and formation of reactive oxygen intermediates are required steps in K+ deprivation-induced apoptosis of these neurons. Here we show that neurotrophins, members of the nerve growth factor gene family, protect from K+/serum deprivation-induced apoptotic death of cerebellar granule neurons in a temporally distinct manner. Switching granule neurons, on day in vitro (DIV) 4, 10, 20, 30, or 40, from high-K+ to low-K+/serum-free medium decreased viability by >50% when measured after 30 h. Treatment of low-K+ granule neurons at DIV 4 with nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotrophin-3, or neurotrophin-4/5 (NT-4/5) demonstrated concentration-dependent (1-100 ng/ml) protective effects only for BDNF and NT-4/5. Between DIV 10 and 20, K+-deprived granule neurons showed decreasing sensitivity to BDNF and no response to NT-4/5. Cerebellar granule neuron death induced by K+ withdrawal at DIV 30 and 40 was blocked only by neurotrophin-3. BDNF and NT-4/5 also circumvented glutamate-induced oxidative death in DIV 1-2 granule neurons. Granule neuron death caused by K+ withdrawal or glutamate-triggered oxidative stress was, moreover, limited by free radical scavengers like melatonin. Neurotrophin-protective effects, but not those of antioxidants, were blocked by selective inhibitors of phosphatidylinositol 3-kinase or the mitogen-activated protein kinase pathway, depending on the nature of the oxidant stress. These observations indicate that the survival-promoting effects of neurotrophins for central neurons, whose cellular antioxidant defenses are challenged, require activation of distinct signal transduction pathways.     

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Bcl-2 family proteins are principal regulators of cell death during normal development as well as in many disease states. Differentiated cerebellar granule neurons are protected from apoptosis by depolarizing concentrations of potassium. Further, these cells acquire resistance to glutamate-mediated excitotoxicity when pre-exposed to subtoxic concentrations of the glutamate receptor agonist, N-methyl-D-aspartate. Here, we report that the expression of bcl-2, bcl-xL, bcl-xS, bax and bad mRNA as well as of Bcl-2, Bax, Bcl-XL, Bcl-XS and Bag-1 proteins is not modulated in these two paradigms of neuronal cell death. However, mitochondrial release of cytochrome c, which is thought to be controlled by Bcl-2 family proteins, is detected 5 h after switching the neurons to low potassium conditions. Thus, there appears to be regulation of Bcl-2 family protein bioactivity in the absence of altered protein expression during potassium deprivation-induced apoptosis of cerebellar granule neurons.     

Lithium protects rat cerebellar granule cells against apoptosis induced by anticonvulsants, phenytoin and carbamazepine

We have studied the neuroprotective actions of lithium against various insults in cultured cerebellar granule cells of rats. The anticonvulsants, phenytoin and carbamazepine, have been shown to induce apoptosis of cerebellar granule cells at high concentrations. Here we found that co-presence of LiCl (1-10 mM) dose-dependently protected against phenytoin (20 microM)- and carbamazepine (100 microM)-induced neuronal apoptosis as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide metabolism, morphological inspection, chromatin condensation and DNA fragmentation. These neuroprotective effects were not prevented by inclusion of myoinositol nor mimicked by a potent inositol monophosphatase inhibitor, suggestive of a mechanism independent of inositol monophosphatase blockade. Lithium also significantly protected against apoptosis of cerebellar granule cells induced by aging of the cultures. Additionally, lithium suppressed death of cerebellar granule cells exposed to a low concentration of extracellular potassium. In contrast, it had no protective effect on cell death induced by Ca++ ionophores, a Na+ channel opener, a protein kinase inhibitor, a nitric oxide donor or H2O2. Thus, lithium has robust neuroprotective effects against apoptotic cell death induced by multiple insults with limited selectivity. These actions provide a new avenue to study the molecular and cellular mechanisms of this drug.     

The dietary excitotoxins beta-N-methylamino-L-alanine and beta-N-oxalylamino-L-alanine induce necrotic- and apoptotic-like death of rat cerebellar granule cells

The neurotoxic properties of the dietary excitotoxins beta-N-methylamino-L-alanine and beta-N-oxalylamino-L-alanine have been studied in rat cerebellar granule cells and compared with those of glutamate. Glutamate caused dose-dependent death of cerebellar granule cells after a 30-min exposure when viability was assessed 24 h later. Beta-N-methylamino-L-alanine and beta-N-oxalylamino-L-alanine, however, were toxic only after 24 or 48 h of exposure. The neurotoxic effects of beta-N-methylamino-L-alanine were blocked by D(-)-2-amino-5-phosphonopentanoic acid, and those of beta-N-oxalylamino-L-alanine were blocked by kynurenic acid, which demonstrated that these excitotoxins caused cerebellar granule cell death through the activation of glutamate receptors. The features of this death were examined morphologically (fluorescent dyes, electron microscopy) and biochemically (conventional agarose gel electrophoresis, effect of aurintricarboxylic acid). Characteristics of apoptosis were identified by transferring cerebellar granule cells from a high K+ (30 mM)- to a low K+ (10 mM)-containing medium. In cerebellar granule cells exposed to beta-N-methylamino-L-alanine or beta-N-oxalylamino-L-alanine (3 mM), hallmarks of necrotic- and apoptotic-like death were observed at various time points over a 72-h period. Therefore, in cerebellar granule cells, beta-N-methylamino-L-alanine and beta-N-oxalylamino-L-alanine induce death over 12-72 h of exposure via a mechanism that involves both necrotic- and apoptotic-like cell death.     

Modulation of expression of glucose transporters GLUT3 and GLUT1 by potassium and N-methyl-D-aspartate in cultured cerebellar granule neurons

Depolarization is known to stimulate neuronal oxidative metabolism. As glucose is the primary fuel for oxidative metabolism in the brain, the entry of glucose into neural cells is a potential control point for any regulatory events in brain metabolism. Therefore, the effects of depolarizing stimuli, high K+ and N-methyl-D-aspartate (NMDA), were examined on the functional expression of glucose transporter isoforms GLUT1 and GLUT3 in primary cultured cerebellar granule neurons. Higher levels of glucose transport activity were observed in neurons cultured in 25 mM KCl (K25) compared to those in 5 and 15 mM KCl (K5 and K15). The elevated glucose transport activity correlated with increased levels of GLUT3 protein and, to a lesser extent, GLUT1. Both GLUT3 and GLUT1 were regulated at the level of mRNA expression. Addition of NMDA to K5 and K15 cultures increased both glucose uptake and GLUT3 protein levels, with smaller changes in GLUT1. NMDA effects were not additive with K25 effects. All these changes were observed only with chronic exposure of neurons to high K+ or NMDA; no acute effects on glucose uptake or transporter expression were found. Thus, chronic depolarization of primary cerebellar granule neurons acts as a stimulus for the expression of the neuronal GLUT3 glucose transporter isoform.     

Acute and chronic changes in K(+)-induced depolarization alter NMDA and nNOS gene expression in cultured cerebellar granule cells

The influence of low or high (10 or 25 mM) K(+)-induced membrane depolarization on the mRNA levels for NMDA receptor subunits was investigated by RNase protection assay in cultured rat cerebellar granule cells. Cells, maintained for 7 days in K25+, a condition that promotes their survival and maturation, express the highest levels of NR-1 and NR-2A mRNA, whereas NR-2B is maximally expressed in cells grown in K10+. Acute changes in medium K+ concentration had a significant effect on the mRNA levels for NMDA receptor subunits. A concomitant reduction of NR-2A mRNA and induction of NR-2B was observed following a 24-h shift of the culture medium from K25+ to K10+. Under these circumstances NR-2C, not detected in basal conditions, became expressed. Neuronal nitric oxide synthase, an enzyme linked to NMDA receptor activation, was also influenced by growth conditions. Its expression, higher under low excitation (K10+), is induced in the shift from K25+ to K10+ and is markedly decreased in the opposite situation. These data indicate that several factors may influence the expression of NMDA receptor subunits and consequently may modulate the function of this receptor complex and its adaptation to acute and chronic changes in neuronal activity.     

Evidence for role of transforming growth factor-beta in RRR-alpha-tocopheryl succinate-induced apoptosis of human MDA-MB-435 breast cancer cells

MDA-MB-435 human breast cancer cells treated with 10 micrograms/ml of RRR-alpha-tocopheryl succinate (vitamin E succinate, VES) for one, two, three, and four days exhibit 9%, 19%, 51%, and 73% apoptotic cells, respectively. Likewise, cells cultured for one, two, and three days with conditioned media (CM) obtained from MDA-MB-435 cells treated with VES exhibit 10%, 36%, and 74% apoptosis, respectively. A quantitative luciferase-based assay showed CM from VES-treated cells collected at 24 and 48 hours after treatment initiation to contain 75 and 32 pg of active transforming growth factor-beta (TGF-beta), respectively, per 10(6) cells. Although purified TGF-beta 1 is not an effective apoptotic agent for MDA-MD-435 cells, cotreatment of the cells for three days with suboptimal levels of VES (2.5 and 5 micrograms/ml) + 10 ng/ml of purified TGF-beta 1 enhanced apoptosis by 66% and 68%, respectively. Interference of the TGF-beta-signaling pathway by transient transfection of MDA-MB-435 cells with antisense oligomers to TGF-beta type II receptor (TGF-beta R-II) blocked VES-induced apoptosis. Likewise, addition of neutralizing antibodies to TGF-beta 1 or to all three mammalian isoforms of TGF-beta (TGF-beta 1, -beta 2, -beta 3) blocked VES- and CM-induced apoptosis. Furthermore, inhibitors of TGF-beta conversion from an inactive latent form to a biologically active form inhibited VES-induced apoptosis. In summary, the ability to reduce apoptosis by blocking TGF-beta or the TGF-beta receptor-signaling pathway with antisense oligomers or ligand-neutralizing antibodies or prevention of activation of TGF-beta indicates a role for TGF-beta signaling in VES-induced apoptosis.     

Brain-derived neurotrophic factor (BDNF) protects cultured rat cerebellar granule neurons against glucose deprivation-induced apoptosis

In the present study, cell death induced by glucose deprivation in primary cultures of cerebellar granule neurons was examined. Glucose deprivation-induced apoptotic cell death was demonstrated using the terminal transferase-mediated (TdT) deoxyuridine triphosphate (d-UTP)-biotin nick end labeling (TUNEL) method and DNA fragmentation assays. When the effects of different neurotrophins on the survival of cerebellar granule neurons after glucose deprivation were assessed, BDNF, but not NT-3 or NGF, was found to protect cerebellar granule neurons against glucose deprivation-induced cell death. In addition, BDNF treatment increased c-Fos immunoreactivity in the cerebellar granule neurons. These results are consistent with the hypothesis that neuronal death due to glucose deprivation has a significant apoptotic component and that neurotrophins can protect against hypoglycemic damage.     

Nitric oxide-dependent production of cGMP supports the survival of rat embryonic motor neurons cultured with brain-derived neurotrophic factor

Trophic factor deprivation induces neuronal nitric oxide synthase (NOS) and apoptosis of rat embryonic motor neurons in culture. We report here that motor neurons constitutively express endothelial NOS that helps support the survival of motor neurons cultured with brain-derived neurotrophic factor (BDNF) by activating the nitric oxide-dependent soluble guanylate cyclase. Exposure of BDNF-treated motor neurons to nitro-L-arginine methyl ester (L-NAME) decreased cell survival 40-50% 24 hr after plating. Both low steady-state concentrations of exogenous nitric oxide (<0.1 microM) and cGMP analogs protected BDNF-treated motor neurons from death induced by L-NAME. Equivalent concentrations of cAMP analogs did not affect cell survival. Inhibition of nitric oxide-sensitive guanylate cyclase with 2 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reduced the survival of BDNF-treated motor neurons by 35%. cGMP analogs also protected from ODQ-induced motor neuron death, whereas exogenous nitric oxide did not. In all cases, cell death was prevented with caspase inhibitors. Our results suggest that nitric oxide-stimulated cGMP synthesis helps to prevent apoptosis in BDNF-treated motor neurons.     

Increased amyloidogenic secretion in cerebellar granule cells undergoing apoptosis

Some clues suggest that neuronal damage induces a secondary change of amyloid beta protein (Abeta) metabolism. We investigated this possibility by analyzing the secretion of Abeta and processing of its precursor protein (amyloid precursor protein, APP) in an in vitro model of neuronal apoptosis. Primary cultures of rat cerebellar granule neurons were metabolically labeled with [35S]methionine. Apoptosis was induced by shifting extracellular KCl concentration from 25 mM to 5 mM for 6 h. Control and apoptotic neurons were then subjected to depolarization-stimulated secretion. Constitutive and stimulated secretion media and cell lysates were immunoprecipitated with antibodies recognizing regions of Abeta, full-length APP, alpha- and beta-APP secreted forms. Immunoprecipitated proteins were separated by SDS/PAGE and quantitated with a PhosphorImager densitometer. Although intracellular full-length APP was not significantly changed after apoptosis, the monomeric and oligomeric forms of 4-kDa Abeta were 3-fold higher in depolarization-stimulated secretion compared with control neurons. Such increments were paralleled by a corresponding increase of the beta-APPs/alpha-APPs ratio in apoptotic secretion. Immunofluorescence studies performed with an antibody recognizing an epitope located in the Abeta sequence showed that the Abeta signal observed in the cytoplasm and in the Golgi apparatus of control neurons is uniformly redistributed in the condensed cytoplasm of apoptotic cells. These studies indicate that neuronal apoptosis is associated with a significant increase of metabolic products derived from beta-secretase cleavage and suggest that an overproduction of Abeta may be the consequence of neuronal damage from various causes.